Sodium absorption in the distal nephron through the epithelial Na+ channel, ENaC, is an important regulator of extracellular fluid volume and blood pressure. Mutations that delete the cytoplasmic C-teminus of the beta and gamma hENaC subunits cause increased Na+ absorption and hypertension (Liddle's syndrome). Loss of function mutations in hENaC cause Na+ wasting (pseudohypoaldosteronism type 1). In preliminary studies, mutations associated with Liddle's syndrome increase Na+ current by increasing the number of channels in the plasma membrane. Mutation of the C-terminal sequence PPPXYXXL in each hENaC subunit reproduced these findings. Interestingly, this sequence is similar to internalization motifs found in a number of proteins. The goal of this application is to understand the function and regulation of hENaC to provide insight into basic mechanisms of Na+ transport and blood pressure control. There are three Specific Aims: 1) To investigate the mechanism(s) of increased surface expression of hENaC caused by Liddle's mutations. The hypothesis to be tested is that the PPPXYXXL motif is important for the internalization of hENaC, and that mutation or deletion of this motif, decreases the rate of channel internalization. An alternate hypothesis, that Liddle's mutations increase the rate of insertion of hENaC into the plasma membrane, will also be tested; 2) Preliminary results show that hENaC function is regulated by the second-messengers cAMP and PKC. The hypothesis to be tested is that these second-messengers regulate hENaC function by altering cell surface expression. An alternate hypothesis, that they alter channel gating, will also be tested; and 3) An important property of ENaC is its high degree of selectivity for Na+ over K+. This is determined by amino aid residues that line the channel pore. Which of the three subunits contribute to the pore will be determined and specific residues that line the pore will be identified, by covalent modification of cysteine residues. Their preliminary results found that two cysteines in the second membrane-spanning segment of gamma ENaC line the channel pore. Using site-directed mutagenesis, they will determine whether analogous residues in the alpha and beta subunits also line the pore. They will also use this strategy to identify other residues in ENaC that line the pore in order to begin to define the structure of the pore, and the molecular basis of its ion selectivity.